One of the most important constraints to soybean production in some parts of the world is soybean rust, caused by Phakopsora pachyrhizi. The lack of high-yielding cultivars with high levels of resistance to the pathogen has prompted a search for novel sources of resistance. Twenty-six soybean germplasm accessions were evaluated for resistance in the greenhouse. Two weeks after inoculation with soybean rust, disease severity was determined. Accessions PI417089B (MG IX), PI459025B (Rpp4; MG VIII), and PI612157 (MG VIII) were found to have more than 50% disease severity, whereas PI567104B, PI615437, PI605773, PI615437, PI605773, and PI567090 showed greater resistance to a 2008 P. pachyrhizi isolate from Quincy, Florida. Significant differences in disease severity were observed among the 26 accessions. Soybean lines from three F5 recombinant inbred line (RIL) populations were also rated for resistance to natural rust infection in the field in 2008 in Quincy. F5:6 progenies from subsets of susceptible and resistant lines from each population were selected for evaluation in the greenhouse to compare their responses with those observed in the field. Significant differences in rust severity and sporulation between the field and greenhouse were observed among lines from the three F5-derived RIL populations. Discrepancies between the field and greenhouse ratings may be indicative of a difference in the genetic composition of the P. pachyrhizi populations or in differences between the reactions of seedlings and adult plants.